THE ASSOCIATION OF LIPID ACTIVATORS WITH THE AMPHIPATHIC HELICAL DOMAIN OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE ACCELERATES CATALYSIS BYINCREASING THE AFFINITY OF THE ENZYME FOR CTP

The biochemical mechanism for the regulation of enzyme activity by lip id modulators and the role of the amphipathic alpha-helical domain of CTP:phosphocholine cytidylyltransferase (CT) was investigated by analy zing the kinetic properties of the wild-type protein and two truncatio n mutants isolated from a baculovirus expression system. The CT[Delta 312-367] mutant protein lacked the carboxyl-terminal phosphorylation d omain and retained high catalytic activity along with both positive an d negative regulation by lipid modulators. The CT[Delta 257-367] delet ion removed in addition the region containing three consecutive amphip athic alpha-helical repeats The CT[Delta 257-367] mutant protein exhib ited a significantly lower specific activity compared to CT or CT[Delt a 312-367] when expressed in either insect or mammalian cells; however , CT[Delta 257-367] activity was refractory to either stimulation or i nhibition by lipid regulators. Lipid activators accelerated CT activit y by decreasing the K-m for CTP from 24.7 mM in their absence to 0.7 m M in their presence. The K-m for phosphocholine was not affected by li pid activators. The activity of CT[Delta 257-367] was comparable to th e activity of wildtype CT in the absence of lipid activators and the C TP K-m for CT[Delta 257-367] was 13.9 mM. The enzymatic properties of the CT[Delta 231-367] mutant were comparable to those exhibited by the CT[257-367] mutant indicating that removal of residues 231 through 25 7 did not have any additional influence on the lipid regulation of the enzyme. Thus, the region between residues 257 and 312 was required to confer lipid regulation on CT, and the association of activating lipi ds with this region of the protein stimulated catalysis by increasing the affinity of the enzyme for CTP.